- Email: [email protected]
Effect of ultrasound on physicochemical and foaming properties of a protein concentrate from giant squid (Dosidicus gigas) mantle
Journal Pre-proof Effect of ultrasound on physicochemical and foaming properties of a protein concentrate from giant squid (Dosidicus gigas) mantle Isabel Arredondo-Parada, Wilfrido Torres-Arreola, Guadalupe M. Suárez-Jiménez, Juan C. Ramírez-Suárez, Josué E. Juárez-Onofre, Francisco Rodríguez-Félix, E. Marquez-Rios PII:
S0023-6438(19)31296-4
DOI:
https://doi.org/10.1016/j.lwt.2019.108954
Reference:
YFSTL 108954
To appear in:
LWT - Food Science and Technology
Received Date: 16 August 2019 Revised Date:
17 November 2019
Accepted Date: 13 December 2019
Please cite this article as: Arredondo-Parada, I., Torres-Arreola, W., Suárez-Jiménez, G.M., RamírezSuárez, J.C., Juárez-Onofre, Josué.E., Rodríguez-Félix, F., Marquez-Rios, E., Effect of ultrasound on physicochemical and foaming properties of a protein concentrate from giant squid (Dosidicus gigas) mantle, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2019.108954. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
UNIVERSIDAD DE SONORA DEPARTAMENTO DE INVESTIGACIÓN Y POSGRADO EN ALIMENTOS
Hermosillo, Sonora, México. November 16, 2019
Rakesh K. Singh Editor-in-Chief LWT-Food Science and Technology
Dear Editor This research has been carried out by the first author of this manuscript, it is the product of his master thesis. The researchers Wilfrido Torres-Arreola, Guadalupe M. Suárez-Jiménez and Juan C. Ramírez-Suárez were the student's thesis advisors. Josué E. Juárez-Onofre supported to the student in the determination of particle size, while Francisco Rodríguez-Félix supported the rheology section. Finally, I was the director of this thesis.
Best regards Enrique Márquez Ríos, PhD. E-mail: [email protected] Main Researcher
1
1
Effect of ultrasound on physicochemical and foaming properties of a protein
2
concentrate from giant squid (Dosidicus gigas) mantle
3 4 5
Isabel Arredondo-Parada1, Wilfrido Torres-Arreola1, Guadalupe M. Suárez-Jiménez1, Juan C. Ramírez-Suárez2, Josué E. Juárez-Onofre3, Francisco Rodríguez-Félix1 and Marquez-Rios E1*
6 7 8 9
1
Departamento de Investigación y Posgrado en Alimentos. Universidad de Sonora. Boulevard Luis Encinas y Rosales, 83000. Hermosillo, Sonora, México.
10 11
2
12 13
3
Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a la Victoria, Km 0.6, 83304 Hermosillo, Sonora, México.
Departamento de Física. Universidad de Sonora. Boulevard Luis Encinas y Rosales, 83000. Hermosillo, Sonora, México.
14 15
*Corresponding author: Enrique Márquez Ríos, PhD
16
E-mail: [email protected]
17 18
2
19
Abstract
20
Giant squid (Dosidicus gigas) proteins have appropriate functional properties, albeit of
21
smaller magnitude in comparison to other marine species. Therefore, this research
22
characterizes the ultrasound-induced (20 kHz; 20 and 40% amplitude; 0, 1, 3 and 5
23
min) changes to the physicochemical and foaming properties of mantle proteins. The
24
changes in pH, electrophoretic profile, viscosity, surface hydrophobicity, particle size
25
and zeta potential, as well as foaming capacity and stability were evaluated. A slight
26
decrease (p ≥ 0.05) in the pH occurred as the ultrasound time increased. While no
27
changes in SDS-PAGE (reducing and non-reducing) were detected, native PAGE
28
revealed new bands. Ultrasound increased the viscosity and surface hydrophobicity,
29
and decreased the particle size and net surface charge. Moreover, foaming capacity
30
was improved and foaming stability was maintained at 100% for 1 h. Therefore, the
31
application of ultrasound represents an alternative to improve the foaming properties.
32 33 34
Keywords: Ultrasound, Squid protein, Functional property, Foaming capacity
3
35
1. Introduction
36
The giant squid (Dosidicus gigas) is the most abundant and largest squid
37
species found in the pelagic zone of the eastern Pacific, from Chile up to the Oregon
38
coast. It is one of the most developed fisheries in the Gulf of California, located in
39
northwestern Mexico, which is of great importance in the states of Baja California Sur
40
and Sonora. The commercial appeal of this resource lies in its great abundance, low
41
cost, low fat content and the white color of its meat, as well as the absence of scales
42
and spines, with the mantle representing the main processed anatomical region. In
43
addition, it is characterized by a high yield (up to 75%, including tentacles, of all its
44
parts after evisceration) and valuable source of high-quality protein, due to its easy
45
digestion and the presence of all essential amino acids. Giant squid proteins have
46
appropriate functional properties, albeit of smaller magnitude in comparison to other
47
marine species (Higuera-Barraza, Del Toro-Sánchez, Ruiz-Cruz & Márquez-Ríos,
48
2016; Márquez-Alvarez et al., 2015; Murrieta-Martínez, Ocaño-Higuera, Suárez-
49
Jiménez & Márquez Ríos, 2015). The myofibrillar proteins (especially actin and
50
myosin) play an important role in these properties (Amiri, Sharifian & Soltanizadeh,
51
2018).
52
Protein functionality in a food system is largely attributed to the complexity of
53
the unique amino acid sequence of the protein. From the technological perspective,
54
proteins fulfill several non-nutritional purposes, such as providing or stabilizing the
55
structure in foods, which includes the ability to form or stabilize foams. Foams are
56
defined as gas-in-liquid dispersions, and their stability depends on the method applied
57
in their formation. Therefore, approaches that improve the functionality of proteins
4
58
attract considerable research attention since maintaining the organoleptic attributes of
59
food depends on the characteristics of these macromolecules (Foegeding & Davis,
60
2011; Higuera-Barraza et al., 2017; O’Sullivan, Park, Beevers, Greenwood & Norton,
61
2017).
62
Physical and chemical methods have been used for modifying proteins to
63
improve their functional properties. However, chemical modifications can be
64
detrimental to the nutritional value of the products and may cause adverse effects on
65
health. Among the numerous physical modification strategies (Singh, Benjakul &
66
Kijroongrojana, 2018), interest in high-intensity ultrasound has increased, as its
67
propagation in biological material induces the compression and decompression of
68
bubbles, which modify the physicochemical properties of the material and improve the
69
quality of various systems (Higuera-Barraza et al., 2017).
70
In recent years, ultrasound has been applied to enhance the foaming
71
properties of several protein sources, such as egg white (Stefanović et al., 2017),
72
chicken meat (Xue et al., 2018), beef (Amiri et al., 2018), wheat (Jambrak, Mason,
73
Lelas, Paniwnyk, & Herceg, 2014) and soy (Morales, Martínez, Ruiz-Henestrosa &
74
Pilosof, 2015). This technology induces conformational changes in the protein
75
structure, causing protein unfolding, in turn, exposing the hydrophilic regions to the
76
aqueous phase, and the hydrophobic regions to the gas phase (Singh et al., 2018).
77
However, the application of ultrasound and its effect on the modification of proteins
78
from marine organisms have been scarcely reported (Higuera-Barraza et al., 2017).
79
Therefore, this research examines the effect of ultrasound on the physicochemical
5
80
properties of a protein concentrate (PC) from giant squid (D. gigas) mantle, with a
81
particular focus on improving its foaming properties.
82
2. Materials and methods
83
2.1. Raw material
84
Frozen (–20 °C) giant squid (D. gigas) was commercially obtained at a local
85
fish market (Alvarez Fish Market, Hermosillo, Sonora, México). The mantles were
86
placed in plastic bags and stored in the laboratory at –20 °C until their utilization.
87 88
2.2. Preparation of the PC
89
Frozen squid mantle was thawed at 4–5 °C for 24 h. Each mantle was
90
considered as a repetition of the experiment; therefore, once the complete mantle
91
was minced, it was mixed with cold distilled water (≤ 4 °C) at a 1:3 mince-to-water
92
ratio. After homogenization at 1000 rpm for 2 min, using a tissue homogenizer (Wisd
93
WiseTis HG-15D, Witeg Labortechnik GmbH, Wertheim, Germany), the homogenate
94
was centrifuged at 12,000 × g, 4 °C for 20 min (Sorvall Biofuge Stratos, Thermo
95
Scientific, Hanau, Germany). The precipitate was regarded as the PC and its protein
96
content was analyzed using the standard AOAC (2005) method. The PC was stored
97
at 4 °C until subsequent analysis.
98 99
2.3. Sonication treatment
100
Protein solutions for each treatment was prepared at a concentration of 5
101
mg/mL, based in preliminary studies and previous research carried out by other
102
authors (Higuera-Barraza et al., 2017; Valdez-Hurtado et al., 2019). For this, 100 mL
6
103
of protein solution were sonicated at 20% (22 W) and 40% (38 W) amplitude for 0, 1,
104
3 and 5 min, using a Branson Digital Sonifier SFX 550 (Branson Ultrasonics
105
Corporation, Danbury, CT, USA) operating at 20 kHz and equipped with a 1.27-cm-
106
diameter titanium probe. During sonication, the samples were maintained in an ice
107
bath, and temperature does not exceed 10 °C.
108 109
2.4. pH measurements
110
The pH of the protein solutions was measured (Woyewoda, Shaw, Ke & Burns,
111
1986) before and after pulsed-sonication at 20 ºC. The pH meter (Mettler Toledo,
112
Leicester, UK) was calibrated against standard buffer solutions of known pH, and the
113
pH values were reported as the average ± standard deviation of three replicates.
114 115
2.5. Electrophoretic profile
116
2.5.1. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
117
PAGE) and reducing SDS-PAGE
118
The electrophoretic profile of each treatment was analyzed by reducing
119
(addition of 2-mercaptoethanol) and non-reducing conditions in polyacrylamide gel,
120
using a discontinuous gel (4% stacking gel, 10% separating gel), according to
121
Laemmli (1970). Proteins samples were heated at 95 °C for 5 min, and 50 µg of
122
protein was loaded into each lane. Electrophoresis was conducted in a Mini-Protean 3
123
Cell system at 95 V. Afterward, the gel was stained with a solution composed of
124
Coomassie brilliant blue R-250 (0.125% w/v), methanol (40% v/v) and acetic acid (7%
125
v/v) and then destained with a solution composed of methanol (50% v/v) and acetic
7
126
acid (10% v/v). Images of the gels were captured and analyzed using a GS-800
127
densitometer (Bio-Rad Laboratory Chemicals, Hercules, CA, USA).
128
2.5.2. Native PAGE
129
To perform the electrophoresis under native conditions, a protocol similar to
130
that described above (section 2.5.1) was followed but sodium dodecyl sulphate and 2-
131
mercaptoethanol were not used. Samples (50 µg protein) were not heat-treated. The
132
same staining and destaining protocols were applied (section 2.5.1).
133 134
2.6. Surface hydrophobicity (So)
135
The So was determined using 1-anilino-8-naphthalene-sulfonate (ANS) as a
136
fluorescence probe, as described previously (Kato & Nakai, 1980). The PC were
137
dissolved in 0.01 M phosphate buffer (pH 7.0) to obtain concentrations of 0.1, 0.2,
138
0.3, 0.4, 0.5 and 1.0 mg mL–1 in a final volume of 3 mL. Then, 30 µL of 8.0 mM ANS
139
(prepared in 0.01 M phosphate buffer, pH 7.0) was added. Relative fluorescence
140
intensity (RFI) was measured using a Cary Eclipse spectrofluorometer (Agilent
141
Technologies, Palo Alto, CA, USA) at wavelengths of 370 nm (excitation) and 490 nm
142
(emission). The initial slope of RFI versus protein concentration (mg mL–1) was
143
calculated by linear regression analysis and used as an index of the protein
144
hydrophobicity.
145 146 147 148
2.7. Viscosity Viscosity measurements were performed on 19 mL (5 mg/mL) of protein solution in the shear rate range of 0.1–450 s
–1
at 25 °C, using an MCR 102
8
149
rheometer (Anton-Paar GmbH, Graz, Austria). Results were expressed as viscosity
150
(Pa.s) versus shear rate (1 s–1) (Murrieta-Martínez et al., 2015).
151 152
2.8. Particle size
153
Particle size was evaluated by dynamic light scattering as described by Gordon
154
and Pilosof (2018), using a Malvern Zetasizer Nano ZS (Malvern Instruments, Ltd.,
155
Malvern, Worcestershire, UK) equipped with a Ne–He laser (633 nm). Measurements
156
were performed at a fixed angle of 173° from 0.6 nm to 6 µm, according to the
157
equipment specifications. Samples of the PC (5 mg mL–1) were diluted 1:100 in Milli-
158
Q water and placed into disposable polystyrene cuvettes (101-QS), at room
159
temperature, and each sample was measured 10 times. Finally, size distribution was
160
plotted as the percentage of the relative intensity of scattered light versus the particle
161
diameter. Mie theory was applied to analyze the raw data using Zetasizer version
162
7.10 software (Malvern Instruments, Ltd.).
163 164
2.9. Zeta (ζ)-potential
165
For determination of the ζ-potential, samples (5 mg mL–1) previously diluted
166
1:100 in Milli-Q water were transferred to capillary cells (DTS 106C; Malvern
167
Instruments, Ltd.) for analysis using the same equipment as mentioned above
168
(section 2.8). A method detailed elsewhere (Arzeni, Pérez & Pilosof, 2015) was
169
applied but modified, such that all measurements were taken at a fixed angle of 17°.
170
Mean values (n = 3) were reported.
171
9
172
2.10. Foaming capacity (FC) and foaming stability (FS)
173
FC was determined based on the method of Coffman and Garcia (1977).
174
Briefly, 50 mL (5 mg mL–1) of protein solution was mixed at high speed for 2 min using
175
a WiseTis HG-15D tissue homogenizer (Wisd Laboratory Instruments, Witeg,
176
Germany) and then, immediately poured into a graduated cylinder. FC (Eq. 1) was
177
calculated by measuring the foam volume at zero time. FS (Eq. 2) was evaluated by
178
recording the foam volume at 1-h intervals for up to 6 h.
179 FC (%) =
Vol. after homogenization – Vol. before homogenization Vol. before homogenization
× 100
(1)
180 FS (%) =
Foam volume after time (t) Initial foam volume
× 100
(2)
181 182
2.11. Statistical analyses
183
This experiment used a factorial design consisting of two main effects
184
(amplitude and duration of sonication) at two (20 and 40%) and four levels (0,0, 1.0,
185
2.5 and 5.0 min) respectively. When required, multiple Tukey comparisons were
186
performed at a significance level of 5% (Cochran & Cox, 1992). Data were analyzed
187
using Jump 5.0.1 (SAS Institute, Cary, NC, USA).
188 189
3. Results and discussion
190
3.1. pH measurements
10
191
A slight but non-significant (p > 0.05) decrease in the pH value was observed
192
as the ultrasound application time increased (Fig. 1), suggesting that the applied
193
treatments were not drastic enough to induce an imbalance in the formation of free
194
hydrogen ions (H+) or the protonation of the water–protein system. This result
195
corroborates that reported by Higuera-Barraza et al. (2017), who applied ultrasound
196
(20 kHz; 20 and 40% amplitude; 30, 60 and 90 s) to a solution of giant squid (D.
197
gigas) protein. Differently, however, Amiri et al. (2018) noticed an increase in the pH
198
of myofibrillar protein from beef muscle (Longissimus dorsi) when the sonication time
199
was progressively extended (20 kHz; 100 and 300 W; 10, 20 and 30 min). Such
200
behavior was attributed to the cavitation phenomenon, which generates a local
201
increase of pressure and temperature at the collapse site of the bubbles. As a result,
202
protein unfolding occurs, and free radicals form that then interact with the side-chains,
203
decreasing the acid groups of proteins.
204 205
3.2. Electrophoresis
206
Electrophoresis identified the main protein bands expected for D. gigas,
207
including the heavy myosin chain (~202.66 kDa), heavy meromyosin (~155.42 kDa),
208
paramyosin (~92.47 kDa), light meromyosin (~68.21 kDa), actin (~41.80 kDa) and
209
tropomyosin (~36.86 kDa) (Fig. 2). Ultrasound did not alter the banding pattern,
210
suggesting that the established conditions of time and amplitude of the sonication
211
treatment were not drastic enough to cause a reduction of the disulfide bridges,
212
rupture of peptide bonds or the formation of aggregates by covalent bonds other than
213
disulfide interactions (Fig. 2 a, b). These same observations have been described by
11
214
previous researchers when examining the effect of ultrasound (20 kHz, 70%
215
amplitude, 30 min) on a protein solution from squid ovary (Loligo formosana) (Singh
216
et al., 2018), and the ultrasonication (20 kHz, 20 and 40% amplitude, 30, 60 and 90 s)
217
of a protein solution of D. gigas (Higuera-Barraza et al., 2017). It should be noted that
218
not finding changes in the banding pattern is expected since this indicates that the
219
treatment is not drastic and does not promote protein hydrolysis or aggregate
220
formation.
221
Regarding the native gel (Fig. 2 c), the main bands reported for this species
222
were observed, namely, myosin (~500 kDa), paramyosin (~220 kDa) and actin (~43–
223
50 kDa) (Tolano-Villaverde et al., 2018) but, also, two new bands appeared below the
224
myosin band, in all treatments, except for the control and 20-1 treatment. It implies
225
that the ultrasound treatments (amplitude and time) promoted the breakdown of
226
electrostatic or Van der Waals interactions between the myosin subunits.
227 228
3.3. Surface hydrophobicity (So)
229
For all treatments, there was an upward trend in the So as time and amplitude
230
increased (Fig. 3), which indicates that the ultrasound modified the three-dimensional
231
structure of the protein. The control sample (without pulses) showed a slope of 98.2
232
(R2 = 0.99), while the sample 20-3 showed the highest value, 116.0 (R2 = 0.99), which
233
corresponds to an increased hydrophobicity of 18.1%. However, sample 20-3 did not
234
show a significant difference (p > 0.05) from the rest of the treatments (20-5, 40-1, 40-
235
3 and 40-5). This increased exposure of hydrophobic residues can be explained by
236
the unfolding of the protein associated with the rupture of non-covalent bonds, as
12
237
shown in the electrophoretic profile, due to the cavitation phenomenon. Similarly,
238
Higuera-Barraza et al. (2017) observed an increase in the So of giant squid (D. gigas)
239
protein solution following exposure to ultrasound (20 kHz, 20 and 40% amplitude, 30,
240
60 and 90 s). In another study, Hu et al. (2013) applied ultrasound (20 kHz; 200, 400
241
and 600 W; 15 and 30 min) to soy protein solution, and detected an increase in So
242
when time and applied energy increased. Instead, when Chandrapala, Zisu, Palmer,
243
Kentish & Ashokkumar (2011) applied ultrasound (20 kHz; 50% amplitude; 1, 5, 10,
244
20, 30 and 60 min) to whey protein solution, So increased only in the first 5 min of
245
sonication, and then tended to decrease.
246 247
3.4. Viscosity
248
As mentioned above (section 3.2 and 3.3), the ultrasound treatment caused
249
conformational changes in the proteins that increased the So and produced two new
250
protein bands on native PAGE. These conformational changes alter the resistance to
251
flow, leading to an increase in viscosity as the amplitude and sonication time
252
increases (Fig. 4 a). The protein unfolding produces conformational changes; it
253
allowed a better interaction of side chains with amino acids of other proteins.
254
Therefore, when a protein is unfolding, it has a greater capacity to interact with water
255
and other proteins, thus enhancing viscosity. However, unlike the control treatment,
256
which showed a Newtonian behavior, the remaining treatments displayed a
257
pseudoplastic behavior. In all treatments, a reduction in viscosity was shown as the
258
shear rate increased. When the shear rate increases to overcome the Brownian
259
motions and breakdown the chemical bonds, the protein strands are aligned parallel
13
260
to the direction of flow, causing less flow resistance and resulting in lower viscosity
261
(Amiri et al., 2018). This phenomenon was evidenced by the control and 20-1
262
treatments, which showed less shear strength as the shear rate increased (Fig. 4 b).
263
These results agree with the findings already discussed in this paper. Namely,
264
the most noticeable changes are particularly evident following the 20-3 and more
265
extreme (20-5, 40-1, 40-3 and 40-5) treatments. The increase in viscosity or stress
266
may be related to the protein unfolding, which allows a greater exposure of
267
hydrophobic and hydrophilic residues to the aqueous environment and, possibly,
268
improving the protein–water–protein interaction. Tan, Chin, Yusof, Taip and Abdullah
269
(2015) evaluated the effect of ultrasound (20 kHz; 20, 40 and 60% amplitude, 5, 15
270
and 25 min) on a whey protein solution and, consistent with the present finding, found
271
an upward trend in viscosity as the time and amplitude were increased. The authors
272
of that study (Tan et al., 2015) attributed this behavior to the cavitation effect, which
273
could have interrupted the electrostatic interactions of the proteins, causing them to
274
unfold and making them less compact, thereby imparting a greater resistance to flow.
275
However, an increase in viscosity due to the application of ultrasound is not always
276
recorded. For instance, in a study conducted by Amiri et al. (2018), the viscosity of
277
the protein solution decreased with the increase in time and energy of the ultrasound
278
(20 kHz; 100 and 300 W; 10, 20 and 30 min). This behavior is related to the physical
279
forces produced during cavitation, which disrupt the interactions between the
280
filaments of the myofibrillar protein and, consequently, a rearrangement of the
281
molecules in the fluid, leading to lower resistance to flow (Amiri et al., 2018).
14
282
From the plot of the viscosity behavior versus time at a constant shear rate, it
283
was possible to see a good stability, irrespective of the treatment (Fig. 4 c) and this
284
might explain the excellent EE of giant squid proteins. According to the results, the
285
increase or decrease of viscosity, as well as its stability over time, will depend on the
286
inherent characteristics of the protein system and the ultrasound conditions applied.
287 288
3.5. Particle size
289
In comparison to the control, a reduction in the particle size occurred, which
290
was most apparent in the treatments performed at 40% amplitude (Fig. 5). It is
291
possible that the amount of energy applied to the system disintegrates any
292
agglomerates, which could affect the viscosity of the system, as previously described
293
(section 3.4). After ultrasonication (20 kHz; 10, 20 and 30 min) of myofibrillar protein
294
solution from beef muscle (L. dorsi), Amiri et al. (2018) found a greater effect on the
295
particle size distribution in the treatments with 300 than 100 W, with a considerable
296
decrease in particle size as the ultrasound time increased. In another study,
297
ultrasound (20 kHz; 0, 60 and 90% amplitude; 20 and 40 min) induced an increase in
298
the particle size and polydispersity index of ovalbumin solution (Xiong et al., 2016),
299
reasoned by the turbulent forces that increased the speed of collision and
300
aggregation,
301
Contrariwise, the particle size of a solution of protein isolate from sunflower meal was
302
decreased by ultrasound (20 kHz; 25% amplitude) applied for 5, 10 and 20 min but
303
when the treatment was extended to 30 min, it increased, indicating that prolonging
forming
unstable
aggregates
through
hydrophobic
interactions.
15
304
the treatment promotes the aggregation of the particles (Malik, Sharma & Saini,
305
2017).
306 307 308
3.6. ζ-potential
309
The ζ-potential reflects the potential difference between the double electric
310
charge of the electrophoretically moving particles and the dispersant layer around
311
them in the sliding plane (Bhattacharjee, 2016). Most proteins have non-polar
312
hydrophobic residues, such as aromatic groups and alkyls, ionic groups, such as –
313
NH3+ and –COO-, as well as hydrophilic polar groups, such as –OH and –NH2, whose
314
balance can influence the surface charge (Martínez-Velasco et al., 2018). All the
315
samples presented a negative net charge (p < 0.05; Table 1). The values are within
316
the threshold of a fine dispersion (–16 to –30 mV), according to the Riddick (1968)
317
scale, which provides information about the tendency of the particles to agglomerate
318
(chemical stability) or remain in suspension (physical stability).
319
Previously, ultrasonication (20 kHz; 0, 60 and 90% amplitude; 20 and 40 min)
320
of ovalbumin decreased the net surface charge, due to a partial unfolding of the
321
protein and the increase in the So, causing a decrease of the electrostatic barrier
322
(Xiong et al., 2016). Likewise, in another study by Xiong et al. (2018), ultrasound (20
323
kHz; 0, 30, 60 and 90% amplitude; 30 min) decreased the surface charge and
324
electrostatic barrier of pea protein isolate, contributing to improving the foaming
325
property. Elsewhere, Jiang et al. (2014) observed an increase in the ζ-potential of
326
black bean protein isolate when ultrasonicated (20 kHz; 12 and 24 min) at 150 and
16
327
300 W, and a decrease when applying 450 W. According to the authors of that work
328
(Jiang et al., 2014), sonication at low and medium power could increase the negative
329
surface charge of proteins due to the electrostatic repulsion between the particles,
330
disrupting protein aggregates and inhibiting aggregation, which leads to an
331
improvement in the protein dispersion stability. In the case of samples in which the ζ-
332
potential decreased, it could be attributed to aggregate formation since a decrease in
333
surface charge could be related to the exposure of hydrophobic apolar residues by
334
the unfolding of the tertiary structure (Jiang et al., 2014).
335 336
3.7. Foaming capacity (FC)
337
The FC was affected by the time and amplitude, observing an increase when
338
compared with the control (Fig. 6). There was a greater effect in the treatments at
339
40% amplitude, with 1 min deemed necessary to improve this property. Meanwhile,
340
when 20% amplitude was used, 3 min was required to achieve the greatest effect.
341
The increase in FC can be attributed to the possible protein unfolding caused by the
342
application of ultrasound, which leads to a greater exposure of hydrophobic regions to
343
the surface, increasing air–protein interactions. These interactions were supported by
344
the So and ζ-potential studies. Singh et al. (2018) enhanced the foaming property of
345
squid ovarian protein (L. formosana) by ultrasound treatment (20 kHz; 30, 40, 50, 60
346
and 70% amplitude; 10, 15, 20, 25 and 30 min). In that instance, 30 min
347
corresponded to the highest FC, whereas, a shorter time gave greater stability. As
348
mentioned by the authors, partial denaturation of the protein can decrease the
349
solubility and induce the formation of aggregates, lowering the foaming ability (Singh
17
350
et al., 2018). Nonetheless, as can be seen here and in the previous literature,
351
ultrasound has the potential to improve the FC.
352 353 354
3.8. Foaming stability (FS)
355
It was observed that the foams developed for all treatments tended to remain
356
stable, even at 6 h after preparation (Fig. 7). The study of this property in protein
357
sources from marine species is scarcely reported in comparison to terrestrial animal
358
proteins. In this sense, Stefanović et al. (2017) showed ultrasound (20 kHz; 40%
359
amplitude) for 2, 5, 10, 15 and 20 min enhanced the FS of a protein solution of egg
360
white, with 15 min corresponding to the most favorable for both FC and FS. It is worth
361
mentioning that the authors made this determination up to 30 min, differing from the
362
present study, in which the remaining foam volume was measurement over 6 h. The
363
foam of the control treatment was maintained for 6 h, while in treatments 20-1, 20-3
364
and 40-1, there was a significant decrease (p < 0.05). Among the treatments studied,
365
20-1, 20-3 and 40-1 showed a higher FC, and so are expected to display the lowest
366
FS, because the larger the foam, generally, the lower the stability. On the contrary,
367
the treatments 20-5, 40-3 and 40-5 presented an FS equal to the control treatment;
368
however, the FC of these treatments was much greater than that of the control. The
369
FS found in this study is outstanding since no research has reported FS for 6 h.
370
Therefore, additional research is required to understand or further explain the
371
excellent FS of the proteins from giant squid mantle.
372
18
373
4. Conclusions
374
The application of ultrasound at 20 kHz at 20 and 40% amplitude positively
375
influenced the functionality of giant squid (D. gigas) protein. This outcome was due to
376
the cavitation effect, which induced a change in the particle size, as well as the three-
377
dimensional structure of the proteins, resulting in greater exposure of hydrophobic
378
groups to the surface, thereby decreasing the net surface charge. In turn, these
379
modifications changed the rheological characteristics, increasing the viscosity and,
380
thereby, the foaming properties. Therefore, ultrasonication under these conditions
381
represents an alternative to improve the foaming property of giant squid mantle
382
proteins.
383 384 385 386
Funding This work was supported by the Consejo Nacional de Ciencia y Tecnología (grant numbers 222150).
387 388
References
389
Amiri, A., Sharifian, P., & Soltanizadeh, N. (2018). Application of ultrasound treatment
390
for improving the physicochemical, functional and rheological properties of
391
myofibrillar proteins. International Journal of Biological Macromolecules, 111,
392
139–147. https://doi.org/10.1016/j.ijbiomac.2017.12.167
393 394 395
AOAC. (2005). Official Methods of Analysis of AOAC International, eighteenth ed., Association of Official Analytical Chemists, Arlington. Arzeni, C., Pérez, O. E., & Pilosof, A. M. R. (2015). Power ultrasound assisted design
19
396
of
397
https://doi.org/10.1007/s11483-015-9407-2
398
egg
albumin
nanoparticles.
Food
Biophysics,
10,
439–446.
Bhattacharjee, S. (2016) DLS and zeta potential – What they are and what they are
399
not?.
Journal
of
Controlled
400
https://doi.org/10.1016/j.jconrel.2016.06.017
Release,
235,
337–351.
401
Chandrapala, J., Zisu, B., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effects
402
of ultrasound on the thermal and structural characteristics of proteins in
403
reconstituted whey protein concentrate. Ultrasonics Sonochemistry, 18, 951–
404
957. https://doi.org/10.1016/j.ultsonch.2010.12.016
405 406
Cochran, W. G., & Cox, G. M. (1992). Experimental Designs. (2nd ed.). New York: John Wiley & Sons.
407
Coffman, C. W., & Garciaj, V. V. (1977). Functional properties and amino acid content
408
of a protein isolate from mung bean flour. International Journal of Food
409
Science
410
2621.1977.tb00132.x
411
and
Technology,
12,
473–484.
https://doi.org/10.1111/j.1365-
Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: A comprehensive
412
approach.
Food
Hydrocolloids,
413
http://dx.doi.org/10.1016/j.foodhyd.2011.05.008
25,
1853–1864.
414
Gordon, L., & Pilosof, A. M. R. (2010). Application of high-intensity ultrasounds to
415
control the size of whey proteins particles. Food Biophysics, 5, 203–210.
416
http://dx.doi.org/10.1007/s11483-010-9161-4
417
Higuera-Barraza, O. A., Del Toro-Sánchez, C. L., Ruiz-Cruz, S., & Márquez-Ríos, E.
418
(2016). Effects of high-energy ultrasound on the functional properties of
20
419
proteins.
Ultrasonics
Sonochemistry,
420
http://dx.doi.org/10.1016/j.ultsonch.2016.02.007
31,
558–562.
421
Higuera-Barraza, O. A., Torres-Arreola, W., Ezquerra-Brauer, J. M., Cinco-
422
Moroyoqui, F. J., Rodríguez-Figueroa, J. C., & Marquez-Ríos, E. (2017). Effect
423
of pulsed ultrasound on the physicochemical characteristics and emulsifying
424
properties
425
Sonochemistry, 38, 829–834. http://dx.doi.org/10.1016/j.ultsonch.2017.01.008
of
squid
(Dosidicus
gigas)
mantle
proteins.
Ultrasonics
426
Hu, H., Wu, J., Li-Chan, E. C. Y., Zhu, L., Zhang, F., Xu, X., Fan, G., Wang, L.,
427
Huang, X., Pan, S. (2013). Effects of ultrasound on structural and physical
428
properties of soy protein isolate (SPI) dispersions. Food Hydrocolloids, 30,
429
647–655. https://doi.org/10.1016/j.foodhyd.2012.08.001
430
Jambrak, A. R., Mason, T. J., Lelas, V., Paniwnyk, L., & Herceg, S. (2014). Effect of
431
ultrasound treatment on particle size and molecular weight of whey proteins.
432
Journal
433
https://doi.org/10.1016/j.jfoodeng.2013.08.012
of
Food
Engineering,
121,
15–23.
434
Jiang, L., Wang, J., Li, Y., Wang, Z., Liang, J., Wang, R., Chen, Y., Ma, W., Qi, B.,
435
Zhang, M. (2014). Effects of ultrasound on the structure and physical
436
properties of black bean protein isolates. Food Research International, 62,
437
595–601. https://doi.org/10.1016/j.foodres.2014.04.022
438
Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe
439
method and its correlation with surface properties of proteins. Biochimica et
440
Biophysica Acta, 624, 13–20. https://doi.org/10.1016/0005-2795(80)90220-2
441
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the
21
442
head of bacteriophage T4. Nature, 227, 680–685.
443
Malik, M. A., Sharma, H. K., & Saini, C. S. (2017). High intensity ultrasound treatment
444
of protein isolate extracted from dephenolized sunflower meal: Effect on
445
physicochemical and functional properties. Ultrasonics Sonochemistry, 39,
446
511– https://doi.org/519. 10.1016/j.ultsonch.2017.05.026
447
Martínez-Velasco, A., Lobato-Calleros, C., Hernández-Rodríguez, B. E., Román-
448
Guerrero, A., Alvarez-Ramirez, J., Vernon-Carter, E. J. (2018). High intensity
449
ultrasound treatment of faba bean (Vicia faba L.) protein: Effect on surface
450
properties, foaming ability and structural changes. Ultrasonics Sonochemistry,
451
44, 97–105. https://doi.org/10.1016/j.ultsonch.2018.02.007
452
Márquez-Alvarez, L. R., Ocano-Higuera, V. M., Rodríguez-Felix, F., Ruiz-Cruz, S.,
453
Del-Toro-Sanchez, C. L., & Márquez-Ríos, E. (2015). Production and
454
functional evaluation of a protein concentrate from giant squid (Dosidicus
455
gigas) fins obtained by alkaline dissolution. Journal of Food Processing and
456
Preservation, 39, 2215–2224. https://doi.org/10.1111/jfpp.12466
457
Morales, R., Martínez, K. D., Ruiz-Henestrosa, V. M. P., & Pilosof, A. M. R. (2015).
458
Modification of foaming properties of soy protein isolate by high ultrasound
459
intensity: Particle size effect. Ultrasonics Sonochemistry, 26, 48–55.
460
https://doi.org/10.1016/j.ultsonch.2015.01.011
461
Murrieta-Martínez, C. L., Ezquerra-Brauer, J. M., Ocaño-Higuera, V. M., Cinco-
462
Moroyoqui, F. J., Torres-Arreola, W., Márquez-Ríos, E. (2015). Isolation and
463
partial characterization of myosin from jumbo squid mantle (Dosidicus gigas),
464
CyTA-Journal
of
Food,
13,
392–399.
22
465
https://doi.org/10.1080/19476337.2014.988183
466
Murrieta-Martínez, C. L., Ocaño-Higuera, V. M., Suárez-Jiménez, G. M., & Márquez
467
Ríos, E. (2016). Squid protein characteristics and their potential industrial
468
applications. Interciencia, 41, 520–525.
469
O’Sullivan, J. J., Park, M., Beevers, J., Greenwood, R. W., & Norton, I. T. (2017)
470
Applications of ultrasound for the functional modification of proteins and
471
nanoemulsion formation: A review. Food Hydrocolloids, 71, 299–310.
472
https://doi.org/10.1016/j.foodhyd.2016.12.037
473 474
Riddick, T. M. (1968). Control of Colloid Stability Through Zeta Potential. Wynnewood: Livingston Pub. Co.
475
Singh, A., Benjakul, S., & Kijroongrojana, K. (2018). Effect of ultrasonication on
476
physicochemical and foaming properties of squid ovary powder. Food
477
Hydrocolloids, 77, 286–296. https://doi.org/10.1016/j.foodhyd.2017.10.005
478
Stefanović, A. B., Jovanović, J. R., Dojčinović, M. B., Lević, S. M., Nedović, V. A.,
479
Bugarski, B. M., & Knežević-Jugović, Z.D. (2017). Effect of the controlled high-
480
intensity ultrasound on improving functionality and structural changes of egg
481
white
482
https://doi.org/10.1007/s11947-017-1884-5
proteins.
Food
Bioprocess
and
Technology,
10,
1224–1239.
483
Tan, M. C., Chin, N. L., Yusof, Y. A., Taip, F. S., Abdullah, J. (2015). Characterization
484
of improved foam aeration and rheological properties of ultrasonically treated
485
whey
486
https://doi.org/10.1016/j.idairyj.2014.09.013
487
protein
suspension.
International
Dairy
Journal,
43,
7–14.
Tolano-Villaverde, I. J., Ocaño-Higuera, V. M., Ezquerra-Brauer, J. M., Santos-
23
488
Sauceda, I., Santacruz-Ortega, H., Cárdenas-López, J. L., Rodríguez-
489
Olibarria, G., & Márquez-Ríos, E. (2018). Physicochemical characterization of
490
actomyosin–paramyosin from giant squid mantle (Dosidicus gigas). Journal of
491
the
492
https://doi.org/10.1002/jsfa.8653
Science
of
Food
and
Agriculture,
98,
1787-1793.
493
Xiong, T., Xiong, W., Ge, M., Xia, J., Li, B., Chen, Y. (2018). Effect of high intensity
494
ultrasound on structure and foaming properties of pea protein isolate. Food
495
Research
496
https://doi.org/10.1016/j.foodres.2018.04.044
International,
109,
260–267.
497
Xiong, W., Wang, Y., Zhang, C., Wan, J., Shah, B. R., Pei, Y., Zhou, B., Li, J., Li, B.
498
(2016). High intensity ultrasound modified ovalbumin: Structure, interface and
499
gelation
500
https://doi.org/10.1016/j.ultsonch.2016.01.014
501
properties.
Ultrasonics
Sonochemistry,
31,
302–309.
Xue, S., Xu, X., Shan, H., Wang, H., Yang, J., & Zhou, G. (2018). Effects of high-
502
intensity
ultrasound,
high-pressure
processing,
and
high-pressure
503
homogenization on the physicochemical and functional properties of
504
myofibrillar proteins. Innovative Food Science and Emerging Technologies, 45,
505
354–360. https://doi.org/10.1016/j.ifset.2017.12.007
506
Woyewoda, A. D., Shaw, S. J., Ke, P. J., & Burns B. G. (1986). Recommended
507
Laboratory Methods for Assessment of Fish Quality, Canadian Technical
508
Report of Fisheries and Aquatic Sciences No. 1448, Nova Scotia, Canada.
509
Table 1. Zeta potential of ultrasound-treated giant squid (Dosidicus gigas) proteins Treatment
Zeta Potential (mV)
C
-25.67a ± 0.84
20-1
-23.03b ± 1.19
20-3
-18.33c ± 0.32
20-5
-18.27c ± 0.75
40-1
-19.43c ± 0.70
40-3
-16.10d ± 0.62
40-5
-19.87c ± 0.83
Control, C; 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. Different superscripts indicate significant differences (p < 0.05). The values are the average of three replicas ± sd. 1 2
Figure captions Figure 1. Ultrasound-induced pH changes to giant squid (Dosidicus gigas) protein. The values are the average of three replicas ± sd. Figure 2. Electrophoretic profile of ultrasound-treated giant squid (Dosidicus gigas) protein under (a) denaturing, (b) reducing, and (c) native conditions. Lane std, marker; C, Control; lanes 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; lanes 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. Figure 3. Surface hydrophobicity (So) of giant squid (Dosidicus gigas) proteins treated by ultrasound. C, Control; 20%-1 min, 20-1; 20%-3 min, 20-3; 20%-5 min, 20-5; 40%-1 min, 40-1; 40%-3 min, 40-3; 40%-5 min, 40-5. The values are the average of three replicas ± sd. Figure 4. Rheological properties of ultrasound-treated giant squid (Dosidicus gigas) proteins: (a) viscosity, (b) shear strength versus shear rate, and (c) viscosity at a constant shear rate. , control; 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. Figure 5. Particle size of ultrasound-treated giant squid (Dosidicus gigas) proteins. C, Control; 20%-1 min, 20-1; 20%-3 min, 20-3; 20%-5 min, 20-5; 40%-1 min, 40-1; 40%-3 min, 40-3; 40%-5 min, 40-5. Figure 6. Foaming capacity of ultrasound-treated giant squid (Dosidicus gigas) proteins. The values are the average of three replicas ± sd. Figure 7. Foaming stability of giant squid (Dosidicus gigas) proteins treated by ultrasound at (a) 20% and (b) 40% amplitude. C, control; 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. The values are the average of three replicas ± sd.
Figure 1
Figure 2a
Figure 2b
Figure 2c
Figure 3
Figure 4a
Figure 4b
Figure 4c
Figure 5
Figure 6
Figure 7a
Figure 7b
Highlights
•
Ultrasound altered the particle size and the 3D structure of the squid proteins
•
The rheological behavior was affected by the ultrasound treatment
•
Ultrasound-induced conformational changes to proteins improved the foaming property
AUTHOR DECLARATION
We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process. He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected].
Dr. Enrique Márquez Ríos, PhD. E-mail: [email protected] Main Researcher
S0023-6438(19)31296-4
DOI:
https://doi.org/10.1016/j.lwt.2019.108954
Reference:
YFSTL 108954
To appear in:
LWT - Food Science and Technology
Received Date: 16 August 2019 Revised Date:
17 November 2019
Accepted Date: 13 December 2019
Please cite this article as: Arredondo-Parada, I., Torres-Arreola, W., Suárez-Jiménez, G.M., RamírezSuárez, J.C., Juárez-Onofre, Josué.E., Rodríguez-Félix, F., Marquez-Rios, E., Effect of ultrasound on physicochemical and foaming properties of a protein concentrate from giant squid (Dosidicus gigas) mantle, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2019.108954. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
UNIVERSIDAD DE SONORA DEPARTAMENTO DE INVESTIGACIÓN Y POSGRADO EN ALIMENTOS
Hermosillo, Sonora, México. November 16, 2019
Rakesh K. Singh Editor-in-Chief LWT-Food Science and Technology
Dear Editor This research has been carried out by the first author of this manuscript, it is the product of his master thesis. The researchers Wilfrido Torres-Arreola, Guadalupe M. Suárez-Jiménez and Juan C. Ramírez-Suárez were the student's thesis advisors. Josué E. Juárez-Onofre supported to the student in the determination of particle size, while Francisco Rodríguez-Félix supported the rheology section. Finally, I was the director of this thesis.
Best regards Enrique Márquez Ríos, PhD. E-mail: [email protected] Main Researcher
1
1
Effect of ultrasound on physicochemical and foaming properties of a protein
2
concentrate from giant squid (Dosidicus gigas) mantle
3 4 5
Isabel Arredondo-Parada1, Wilfrido Torres-Arreola1, Guadalupe M. Suárez-Jiménez1, Juan C. Ramírez-Suárez2, Josué E. Juárez-Onofre3, Francisco Rodríguez-Félix1 and Marquez-Rios E1*
6 7 8 9
1
Departamento de Investigación y Posgrado en Alimentos. Universidad de Sonora. Boulevard Luis Encinas y Rosales, 83000. Hermosillo, Sonora, México.
10 11
2
12 13
3
Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a la Victoria, Km 0.6, 83304 Hermosillo, Sonora, México.
Departamento de Física. Universidad de Sonora. Boulevard Luis Encinas y Rosales, 83000. Hermosillo, Sonora, México.
14 15
*Corresponding author: Enrique Márquez Ríos, PhD
16
E-mail: [email protected]
17 18
2
19
Abstract
20
Giant squid (Dosidicus gigas) proteins have appropriate functional properties, albeit of
21
smaller magnitude in comparison to other marine species. Therefore, this research
22
characterizes the ultrasound-induced (20 kHz; 20 and 40% amplitude; 0, 1, 3 and 5
23
min) changes to the physicochemical and foaming properties of mantle proteins. The
24
changes in pH, electrophoretic profile, viscosity, surface hydrophobicity, particle size
25
and zeta potential, as well as foaming capacity and stability were evaluated. A slight
26
decrease (p ≥ 0.05) in the pH occurred as the ultrasound time increased. While no
27
changes in SDS-PAGE (reducing and non-reducing) were detected, native PAGE
28
revealed new bands. Ultrasound increased the viscosity and surface hydrophobicity,
29
and decreased the particle size and net surface charge. Moreover, foaming capacity
30
was improved and foaming stability was maintained at 100% for 1 h. Therefore, the
31
application of ultrasound represents an alternative to improve the foaming properties.
32 33 34
Keywords: Ultrasound, Squid protein, Functional property, Foaming capacity
3
35
1. Introduction
36
The giant squid (Dosidicus gigas) is the most abundant and largest squid
37
species found in the pelagic zone of the eastern Pacific, from Chile up to the Oregon
38
coast. It is one of the most developed fisheries in the Gulf of California, located in
39
northwestern Mexico, which is of great importance in the states of Baja California Sur
40
and Sonora. The commercial appeal of this resource lies in its great abundance, low
41
cost, low fat content and the white color of its meat, as well as the absence of scales
42
and spines, with the mantle representing the main processed anatomical region. In
43
addition, it is characterized by a high yield (up to 75%, including tentacles, of all its
44
parts after evisceration) and valuable source of high-quality protein, due to its easy
45
digestion and the presence of all essential amino acids. Giant squid proteins have
46
appropriate functional properties, albeit of smaller magnitude in comparison to other
47
marine species (Higuera-Barraza, Del Toro-Sánchez, Ruiz-Cruz & Márquez-Ríos,
48
2016; Márquez-Alvarez et al., 2015; Murrieta-Martínez, Ocaño-Higuera, Suárez-
49
Jiménez & Márquez Ríos, 2015). The myofibrillar proteins (especially actin and
50
myosin) play an important role in these properties (Amiri, Sharifian & Soltanizadeh,
51
2018).
52
Protein functionality in a food system is largely attributed to the complexity of
53
the unique amino acid sequence of the protein. From the technological perspective,
54
proteins fulfill several non-nutritional purposes, such as providing or stabilizing the
55
structure in foods, which includes the ability to form or stabilize foams. Foams are
56
defined as gas-in-liquid dispersions, and their stability depends on the method applied
57
in their formation. Therefore, approaches that improve the functionality of proteins
4
58
attract considerable research attention since maintaining the organoleptic attributes of
59
food depends on the characteristics of these macromolecules (Foegeding & Davis,
60
2011; Higuera-Barraza et al., 2017; O’Sullivan, Park, Beevers, Greenwood & Norton,
61
2017).
62
Physical and chemical methods have been used for modifying proteins to
63
improve their functional properties. However, chemical modifications can be
64
detrimental to the nutritional value of the products and may cause adverse effects on
65
health. Among the numerous physical modification strategies (Singh, Benjakul &
66
Kijroongrojana, 2018), interest in high-intensity ultrasound has increased, as its
67
propagation in biological material induces the compression and decompression of
68
bubbles, which modify the physicochemical properties of the material and improve the
69
quality of various systems (Higuera-Barraza et al., 2017).
70
In recent years, ultrasound has been applied to enhance the foaming
71
properties of several protein sources, such as egg white (Stefanović et al., 2017),
72
chicken meat (Xue et al., 2018), beef (Amiri et al., 2018), wheat (Jambrak, Mason,
73
Lelas, Paniwnyk, & Herceg, 2014) and soy (Morales, Martínez, Ruiz-Henestrosa &
74
Pilosof, 2015). This technology induces conformational changes in the protein
75
structure, causing protein unfolding, in turn, exposing the hydrophilic regions to the
76
aqueous phase, and the hydrophobic regions to the gas phase (Singh et al., 2018).
77
However, the application of ultrasound and its effect on the modification of proteins
78
from marine organisms have been scarcely reported (Higuera-Barraza et al., 2017).
79
Therefore, this research examines the effect of ultrasound on the physicochemical
5
80
properties of a protein concentrate (PC) from giant squid (D. gigas) mantle, with a
81
particular focus on improving its foaming properties.
82
2. Materials and methods
83
2.1. Raw material
84
Frozen (–20 °C) giant squid (D. gigas) was commercially obtained at a local
85
fish market (Alvarez Fish Market, Hermosillo, Sonora, México). The mantles were
86
placed in plastic bags and stored in the laboratory at –20 °C until their utilization.
87 88
2.2. Preparation of the PC
89
Frozen squid mantle was thawed at 4–5 °C for 24 h. Each mantle was
90
considered as a repetition of the experiment; therefore, once the complete mantle
91
was minced, it was mixed with cold distilled water (≤ 4 °C) at a 1:3 mince-to-water
92
ratio. After homogenization at 1000 rpm for 2 min, using a tissue homogenizer (Wisd
93
WiseTis HG-15D, Witeg Labortechnik GmbH, Wertheim, Germany), the homogenate
94
was centrifuged at 12,000 × g, 4 °C for 20 min (Sorvall Biofuge Stratos, Thermo
95
Scientific, Hanau, Germany). The precipitate was regarded as the PC and its protein
96
content was analyzed using the standard AOAC (2005) method. The PC was stored
97
at 4 °C until subsequent analysis.
98 99
2.3. Sonication treatment
100
Protein solutions for each treatment was prepared at a concentration of 5
101
mg/mL, based in preliminary studies and previous research carried out by other
102
authors (Higuera-Barraza et al., 2017; Valdez-Hurtado et al., 2019). For this, 100 mL
6
103
of protein solution were sonicated at 20% (22 W) and 40% (38 W) amplitude for 0, 1,
104
3 and 5 min, using a Branson Digital Sonifier SFX 550 (Branson Ultrasonics
105
Corporation, Danbury, CT, USA) operating at 20 kHz and equipped with a 1.27-cm-
106
diameter titanium probe. During sonication, the samples were maintained in an ice
107
bath, and temperature does not exceed 10 °C.
108 109
2.4. pH measurements
110
The pH of the protein solutions was measured (Woyewoda, Shaw, Ke & Burns,
111
1986) before and after pulsed-sonication at 20 ºC. The pH meter (Mettler Toledo,
112
Leicester, UK) was calibrated against standard buffer solutions of known pH, and the
113
pH values were reported as the average ± standard deviation of three replicates.
114 115
2.5. Electrophoretic profile
116
2.5.1. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
117
PAGE) and reducing SDS-PAGE
118
The electrophoretic profile of each treatment was analyzed by reducing
119
(addition of 2-mercaptoethanol) and non-reducing conditions in polyacrylamide gel,
120
using a discontinuous gel (4% stacking gel, 10% separating gel), according to
121
Laemmli (1970). Proteins samples were heated at 95 °C for 5 min, and 50 µg of
122
protein was loaded into each lane. Electrophoresis was conducted in a Mini-Protean 3
123
Cell system at 95 V. Afterward, the gel was stained with a solution composed of
124
Coomassie brilliant blue R-250 (0.125% w/v), methanol (40% v/v) and acetic acid (7%
125
v/v) and then destained with a solution composed of methanol (50% v/v) and acetic
7
126
acid (10% v/v). Images of the gels were captured and analyzed using a GS-800
127
densitometer (Bio-Rad Laboratory Chemicals, Hercules, CA, USA).
128
2.5.2. Native PAGE
129
To perform the electrophoresis under native conditions, a protocol similar to
130
that described above (section 2.5.1) was followed but sodium dodecyl sulphate and 2-
131
mercaptoethanol were not used. Samples (50 µg protein) were not heat-treated. The
132
same staining and destaining protocols were applied (section 2.5.1).
133 134
2.6. Surface hydrophobicity (So)
135
The So was determined using 1-anilino-8-naphthalene-sulfonate (ANS) as a
136
fluorescence probe, as described previously (Kato & Nakai, 1980). The PC were
137
dissolved in 0.01 M phosphate buffer (pH 7.0) to obtain concentrations of 0.1, 0.2,
138
0.3, 0.4, 0.5 and 1.0 mg mL–1 in a final volume of 3 mL. Then, 30 µL of 8.0 mM ANS
139
(prepared in 0.01 M phosphate buffer, pH 7.0) was added. Relative fluorescence
140
intensity (RFI) was measured using a Cary Eclipse spectrofluorometer (Agilent
141
Technologies, Palo Alto, CA, USA) at wavelengths of 370 nm (excitation) and 490 nm
142
(emission). The initial slope of RFI versus protein concentration (mg mL–1) was
143
calculated by linear regression analysis and used as an index of the protein
144
hydrophobicity.
145 146 147 148
2.7. Viscosity Viscosity measurements were performed on 19 mL (5 mg/mL) of protein solution in the shear rate range of 0.1–450 s
–1
at 25 °C, using an MCR 102
8
149
rheometer (Anton-Paar GmbH, Graz, Austria). Results were expressed as viscosity
150
(Pa.s) versus shear rate (1 s–1) (Murrieta-Martínez et al., 2015).
151 152
2.8. Particle size
153
Particle size was evaluated by dynamic light scattering as described by Gordon
154
and Pilosof (2018), using a Malvern Zetasizer Nano ZS (Malvern Instruments, Ltd.,
155
Malvern, Worcestershire, UK) equipped with a Ne–He laser (633 nm). Measurements
156
were performed at a fixed angle of 173° from 0.6 nm to 6 µm, according to the
157
equipment specifications. Samples of the PC (5 mg mL–1) were diluted 1:100 in Milli-
158
Q water and placed into disposable polystyrene cuvettes (101-QS), at room
159
temperature, and each sample was measured 10 times. Finally, size distribution was
160
plotted as the percentage of the relative intensity of scattered light versus the particle
161
diameter. Mie theory was applied to analyze the raw data using Zetasizer version
162
7.10 software (Malvern Instruments, Ltd.).
163 164
2.9. Zeta (ζ)-potential
165
For determination of the ζ-potential, samples (5 mg mL–1) previously diluted
166
1:100 in Milli-Q water were transferred to capillary cells (DTS 106C; Malvern
167
Instruments, Ltd.) for analysis using the same equipment as mentioned above
168
(section 2.8). A method detailed elsewhere (Arzeni, Pérez & Pilosof, 2015) was
169
applied but modified, such that all measurements were taken at a fixed angle of 17°.
170
Mean values (n = 3) were reported.
171
9
172
2.10. Foaming capacity (FC) and foaming stability (FS)
173
FC was determined based on the method of Coffman and Garcia (1977).
174
Briefly, 50 mL (5 mg mL–1) of protein solution was mixed at high speed for 2 min using
175
a WiseTis HG-15D tissue homogenizer (Wisd Laboratory Instruments, Witeg,
176
Germany) and then, immediately poured into a graduated cylinder. FC (Eq. 1) was
177
calculated by measuring the foam volume at zero time. FS (Eq. 2) was evaluated by
178
recording the foam volume at 1-h intervals for up to 6 h.
179 FC (%) =
Vol. after homogenization – Vol. before homogenization Vol. before homogenization
× 100
(1)
180 FS (%) =
Foam volume after time (t) Initial foam volume
× 100
(2)
181 182
2.11. Statistical analyses
183
This experiment used a factorial design consisting of two main effects
184
(amplitude and duration of sonication) at two (20 and 40%) and four levels (0,0, 1.0,
185
2.5 and 5.0 min) respectively. When required, multiple Tukey comparisons were
186
performed at a significance level of 5% (Cochran & Cox, 1992). Data were analyzed
187
using Jump 5.0.1 (SAS Institute, Cary, NC, USA).
188 189
3. Results and discussion
190
3.1. pH measurements
10
191
A slight but non-significant (p > 0.05) decrease in the pH value was observed
192
as the ultrasound application time increased (Fig. 1), suggesting that the applied
193
treatments were not drastic enough to induce an imbalance in the formation of free
194
hydrogen ions (H+) or the protonation of the water–protein system. This result
195
corroborates that reported by Higuera-Barraza et al. (2017), who applied ultrasound
196
(20 kHz; 20 and 40% amplitude; 30, 60 and 90 s) to a solution of giant squid (D.
197
gigas) protein. Differently, however, Amiri et al. (2018) noticed an increase in the pH
198
of myofibrillar protein from beef muscle (Longissimus dorsi) when the sonication time
199
was progressively extended (20 kHz; 100 and 300 W; 10, 20 and 30 min). Such
200
behavior was attributed to the cavitation phenomenon, which generates a local
201
increase of pressure and temperature at the collapse site of the bubbles. As a result,
202
protein unfolding occurs, and free radicals form that then interact with the side-chains,
203
decreasing the acid groups of proteins.
204 205
3.2. Electrophoresis
206
Electrophoresis identified the main protein bands expected for D. gigas,
207
including the heavy myosin chain (~202.66 kDa), heavy meromyosin (~155.42 kDa),
208
paramyosin (~92.47 kDa), light meromyosin (~68.21 kDa), actin (~41.80 kDa) and
209
tropomyosin (~36.86 kDa) (Fig. 2). Ultrasound did not alter the banding pattern,
210
suggesting that the established conditions of time and amplitude of the sonication
211
treatment were not drastic enough to cause a reduction of the disulfide bridges,
212
rupture of peptide bonds or the formation of aggregates by covalent bonds other than
213
disulfide interactions (Fig. 2 a, b). These same observations have been described by
11
214
previous researchers when examining the effect of ultrasound (20 kHz, 70%
215
amplitude, 30 min) on a protein solution from squid ovary (Loligo formosana) (Singh
216
et al., 2018), and the ultrasonication (20 kHz, 20 and 40% amplitude, 30, 60 and 90 s)
217
of a protein solution of D. gigas (Higuera-Barraza et al., 2017). It should be noted that
218
not finding changes in the banding pattern is expected since this indicates that the
219
treatment is not drastic and does not promote protein hydrolysis or aggregate
220
formation.
221
Regarding the native gel (Fig. 2 c), the main bands reported for this species
222
were observed, namely, myosin (~500 kDa), paramyosin (~220 kDa) and actin (~43–
223
50 kDa) (Tolano-Villaverde et al., 2018) but, also, two new bands appeared below the
224
myosin band, in all treatments, except for the control and 20-1 treatment. It implies
225
that the ultrasound treatments (amplitude and time) promoted the breakdown of
226
electrostatic or Van der Waals interactions between the myosin subunits.
227 228
3.3. Surface hydrophobicity (So)
229
For all treatments, there was an upward trend in the So as time and amplitude
230
increased (Fig. 3), which indicates that the ultrasound modified the three-dimensional
231
structure of the protein. The control sample (without pulses) showed a slope of 98.2
232
(R2 = 0.99), while the sample 20-3 showed the highest value, 116.0 (R2 = 0.99), which
233
corresponds to an increased hydrophobicity of 18.1%. However, sample 20-3 did not
234
show a significant difference (p > 0.05) from the rest of the treatments (20-5, 40-1, 40-
235
3 and 40-5). This increased exposure of hydrophobic residues can be explained by
236
the unfolding of the protein associated with the rupture of non-covalent bonds, as
12
237
shown in the electrophoretic profile, due to the cavitation phenomenon. Similarly,
238
Higuera-Barraza et al. (2017) observed an increase in the So of giant squid (D. gigas)
239
protein solution following exposure to ultrasound (20 kHz, 20 and 40% amplitude, 30,
240
60 and 90 s). In another study, Hu et al. (2013) applied ultrasound (20 kHz; 200, 400
241
and 600 W; 15 and 30 min) to soy protein solution, and detected an increase in So
242
when time and applied energy increased. Instead, when Chandrapala, Zisu, Palmer,
243
Kentish & Ashokkumar (2011) applied ultrasound (20 kHz; 50% amplitude; 1, 5, 10,
244
20, 30 and 60 min) to whey protein solution, So increased only in the first 5 min of
245
sonication, and then tended to decrease.
246 247
3.4. Viscosity
248
As mentioned above (section 3.2 and 3.3), the ultrasound treatment caused
249
conformational changes in the proteins that increased the So and produced two new
250
protein bands on native PAGE. These conformational changes alter the resistance to
251
flow, leading to an increase in viscosity as the amplitude and sonication time
252
increases (Fig. 4 a). The protein unfolding produces conformational changes; it
253
allowed a better interaction of side chains with amino acids of other proteins.
254
Therefore, when a protein is unfolding, it has a greater capacity to interact with water
255
and other proteins, thus enhancing viscosity. However, unlike the control treatment,
256
which showed a Newtonian behavior, the remaining treatments displayed a
257
pseudoplastic behavior. In all treatments, a reduction in viscosity was shown as the
258
shear rate increased. When the shear rate increases to overcome the Brownian
259
motions and breakdown the chemical bonds, the protein strands are aligned parallel
13
260
to the direction of flow, causing less flow resistance and resulting in lower viscosity
261
(Amiri et al., 2018). This phenomenon was evidenced by the control and 20-1
262
treatments, which showed less shear strength as the shear rate increased (Fig. 4 b).
263
These results agree with the findings already discussed in this paper. Namely,
264
the most noticeable changes are particularly evident following the 20-3 and more
265
extreme (20-5, 40-1, 40-3 and 40-5) treatments. The increase in viscosity or stress
266
may be related to the protein unfolding, which allows a greater exposure of
267
hydrophobic and hydrophilic residues to the aqueous environment and, possibly,
268
improving the protein–water–protein interaction. Tan, Chin, Yusof, Taip and Abdullah
269
(2015) evaluated the effect of ultrasound (20 kHz; 20, 40 and 60% amplitude, 5, 15
270
and 25 min) on a whey protein solution and, consistent with the present finding, found
271
an upward trend in viscosity as the time and amplitude were increased. The authors
272
of that study (Tan et al., 2015) attributed this behavior to the cavitation effect, which
273
could have interrupted the electrostatic interactions of the proteins, causing them to
274
unfold and making them less compact, thereby imparting a greater resistance to flow.
275
However, an increase in viscosity due to the application of ultrasound is not always
276
recorded. For instance, in a study conducted by Amiri et al. (2018), the viscosity of
277
the protein solution decreased with the increase in time and energy of the ultrasound
278
(20 kHz; 100 and 300 W; 10, 20 and 30 min). This behavior is related to the physical
279
forces produced during cavitation, which disrupt the interactions between the
280
filaments of the myofibrillar protein and, consequently, a rearrangement of the
281
molecules in the fluid, leading to lower resistance to flow (Amiri et al., 2018).
14
282
From the plot of the viscosity behavior versus time at a constant shear rate, it
283
was possible to see a good stability, irrespective of the treatment (Fig. 4 c) and this
284
might explain the excellent EE of giant squid proteins. According to the results, the
285
increase or decrease of viscosity, as well as its stability over time, will depend on the
286
inherent characteristics of the protein system and the ultrasound conditions applied.
287 288
3.5. Particle size
289
In comparison to the control, a reduction in the particle size occurred, which
290
was most apparent in the treatments performed at 40% amplitude (Fig. 5). It is
291
possible that the amount of energy applied to the system disintegrates any
292
agglomerates, which could affect the viscosity of the system, as previously described
293
(section 3.4). After ultrasonication (20 kHz; 10, 20 and 30 min) of myofibrillar protein
294
solution from beef muscle (L. dorsi), Amiri et al. (2018) found a greater effect on the
295
particle size distribution in the treatments with 300 than 100 W, with a considerable
296
decrease in particle size as the ultrasound time increased. In another study,
297
ultrasound (20 kHz; 0, 60 and 90% amplitude; 20 and 40 min) induced an increase in
298
the particle size and polydispersity index of ovalbumin solution (Xiong et al., 2016),
299
reasoned by the turbulent forces that increased the speed of collision and
300
aggregation,
301
Contrariwise, the particle size of a solution of protein isolate from sunflower meal was
302
decreased by ultrasound (20 kHz; 25% amplitude) applied for 5, 10 and 20 min but
303
when the treatment was extended to 30 min, it increased, indicating that prolonging
forming
unstable
aggregates
through
hydrophobic
interactions.
15
304
the treatment promotes the aggregation of the particles (Malik, Sharma & Saini,
305
2017).
306 307 308
3.6. ζ-potential
309
The ζ-potential reflects the potential difference between the double electric
310
charge of the electrophoretically moving particles and the dispersant layer around
311
them in the sliding plane (Bhattacharjee, 2016). Most proteins have non-polar
312
hydrophobic residues, such as aromatic groups and alkyls, ionic groups, such as –
313
NH3+ and –COO-, as well as hydrophilic polar groups, such as –OH and –NH2, whose
314
balance can influence the surface charge (Martínez-Velasco et al., 2018). All the
315
samples presented a negative net charge (p < 0.05; Table 1). The values are within
316
the threshold of a fine dispersion (–16 to –30 mV), according to the Riddick (1968)
317
scale, which provides information about the tendency of the particles to agglomerate
318
(chemical stability) or remain in suspension (physical stability).
319
Previously, ultrasonication (20 kHz; 0, 60 and 90% amplitude; 20 and 40 min)
320
of ovalbumin decreased the net surface charge, due to a partial unfolding of the
321
protein and the increase in the So, causing a decrease of the electrostatic barrier
322
(Xiong et al., 2016). Likewise, in another study by Xiong et al. (2018), ultrasound (20
323
kHz; 0, 30, 60 and 90% amplitude; 30 min) decreased the surface charge and
324
electrostatic barrier of pea protein isolate, contributing to improving the foaming
325
property. Elsewhere, Jiang et al. (2014) observed an increase in the ζ-potential of
326
black bean protein isolate when ultrasonicated (20 kHz; 12 and 24 min) at 150 and
16
327
300 W, and a decrease when applying 450 W. According to the authors of that work
328
(Jiang et al., 2014), sonication at low and medium power could increase the negative
329
surface charge of proteins due to the electrostatic repulsion between the particles,
330
disrupting protein aggregates and inhibiting aggregation, which leads to an
331
improvement in the protein dispersion stability. In the case of samples in which the ζ-
332
potential decreased, it could be attributed to aggregate formation since a decrease in
333
surface charge could be related to the exposure of hydrophobic apolar residues by
334
the unfolding of the tertiary structure (Jiang et al., 2014).
335 336
3.7. Foaming capacity (FC)
337
The FC was affected by the time and amplitude, observing an increase when
338
compared with the control (Fig. 6). There was a greater effect in the treatments at
339
40% amplitude, with 1 min deemed necessary to improve this property. Meanwhile,
340
when 20% amplitude was used, 3 min was required to achieve the greatest effect.
341
The increase in FC can be attributed to the possible protein unfolding caused by the
342
application of ultrasound, which leads to a greater exposure of hydrophobic regions to
343
the surface, increasing air–protein interactions. These interactions were supported by
344
the So and ζ-potential studies. Singh et al. (2018) enhanced the foaming property of
345
squid ovarian protein (L. formosana) by ultrasound treatment (20 kHz; 30, 40, 50, 60
346
and 70% amplitude; 10, 15, 20, 25 and 30 min). In that instance, 30 min
347
corresponded to the highest FC, whereas, a shorter time gave greater stability. As
348
mentioned by the authors, partial denaturation of the protein can decrease the
349
solubility and induce the formation of aggregates, lowering the foaming ability (Singh
17
350
et al., 2018). Nonetheless, as can be seen here and in the previous literature,
351
ultrasound has the potential to improve the FC.
352 353 354
3.8. Foaming stability (FS)
355
It was observed that the foams developed for all treatments tended to remain
356
stable, even at 6 h after preparation (Fig. 7). The study of this property in protein
357
sources from marine species is scarcely reported in comparison to terrestrial animal
358
proteins. In this sense, Stefanović et al. (2017) showed ultrasound (20 kHz; 40%
359
amplitude) for 2, 5, 10, 15 and 20 min enhanced the FS of a protein solution of egg
360
white, with 15 min corresponding to the most favorable for both FC and FS. It is worth
361
mentioning that the authors made this determination up to 30 min, differing from the
362
present study, in which the remaining foam volume was measurement over 6 h. The
363
foam of the control treatment was maintained for 6 h, while in treatments 20-1, 20-3
364
and 40-1, there was a significant decrease (p < 0.05). Among the treatments studied,
365
20-1, 20-3 and 40-1 showed a higher FC, and so are expected to display the lowest
366
FS, because the larger the foam, generally, the lower the stability. On the contrary,
367
the treatments 20-5, 40-3 and 40-5 presented an FS equal to the control treatment;
368
however, the FC of these treatments was much greater than that of the control. The
369
FS found in this study is outstanding since no research has reported FS for 6 h.
370
Therefore, additional research is required to understand or further explain the
371
excellent FS of the proteins from giant squid mantle.
372
18
373
4. Conclusions
374
The application of ultrasound at 20 kHz at 20 and 40% amplitude positively
375
influenced the functionality of giant squid (D. gigas) protein. This outcome was due to
376
the cavitation effect, which induced a change in the particle size, as well as the three-
377
dimensional structure of the proteins, resulting in greater exposure of hydrophobic
378
groups to the surface, thereby decreasing the net surface charge. In turn, these
379
modifications changed the rheological characteristics, increasing the viscosity and,
380
thereby, the foaming properties. Therefore, ultrasonication under these conditions
381
represents an alternative to improve the foaming property of giant squid mantle
382
proteins.
383 384 385 386
Funding This work was supported by the Consejo Nacional de Ciencia y Tecnología (grant numbers 222150).
387 388
References
389
Amiri, A., Sharifian, P., & Soltanizadeh, N. (2018). Application of ultrasound treatment
390
for improving the physicochemical, functional and rheological properties of
391
myofibrillar proteins. International Journal of Biological Macromolecules, 111,
392
139–147. https://doi.org/10.1016/j.ijbiomac.2017.12.167
393 394 395
AOAC. (2005). Official Methods of Analysis of AOAC International, eighteenth ed., Association of Official Analytical Chemists, Arlington. Arzeni, C., Pérez, O. E., & Pilosof, A. M. R. (2015). Power ultrasound assisted design
19
396
of
397
https://doi.org/10.1007/s11483-015-9407-2
398
egg
albumin
nanoparticles.
Food
Biophysics,
10,
439–446.
Bhattacharjee, S. (2016) DLS and zeta potential – What they are and what they are
399
not?.
Journal
of
Controlled
400
https://doi.org/10.1016/j.jconrel.2016.06.017
Release,
235,
337–351.
401
Chandrapala, J., Zisu, B., Palmer, M., Kentish, S., & Ashokkumar, M. (2011). Effects
402
of ultrasound on the thermal and structural characteristics of proteins in
403
reconstituted whey protein concentrate. Ultrasonics Sonochemistry, 18, 951–
404
957. https://doi.org/10.1016/j.ultsonch.2010.12.016
405 406
Cochran, W. G., & Cox, G. M. (1992). Experimental Designs. (2nd ed.). New York: John Wiley & Sons.
407
Coffman, C. W., & Garciaj, V. V. (1977). Functional properties and amino acid content
408
of a protein isolate from mung bean flour. International Journal of Food
409
Science
410
2621.1977.tb00132.x
411
and
Technology,
12,
473–484.
https://doi.org/10.1111/j.1365-
Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: A comprehensive
412
approach.
Food
Hydrocolloids,
413
http://dx.doi.org/10.1016/j.foodhyd.2011.05.008
25,
1853–1864.
414
Gordon, L., & Pilosof, A. M. R. (2010). Application of high-intensity ultrasounds to
415
control the size of whey proteins particles. Food Biophysics, 5, 203–210.
416
http://dx.doi.org/10.1007/s11483-010-9161-4
417
Higuera-Barraza, O. A., Del Toro-Sánchez, C. L., Ruiz-Cruz, S., & Márquez-Ríos, E.
418
(2016). Effects of high-energy ultrasound on the functional properties of
20
419
proteins.
Ultrasonics
Sonochemistry,
420
http://dx.doi.org/10.1016/j.ultsonch.2016.02.007
31,
558–562.
421
Higuera-Barraza, O. A., Torres-Arreola, W., Ezquerra-Brauer, J. M., Cinco-
422
Moroyoqui, F. J., Rodríguez-Figueroa, J. C., & Marquez-Ríos, E. (2017). Effect
423
of pulsed ultrasound on the physicochemical characteristics and emulsifying
424
properties
425
Sonochemistry, 38, 829–834. http://dx.doi.org/10.1016/j.ultsonch.2017.01.008
of
squid
(Dosidicus
gigas)
mantle
proteins.
Ultrasonics
426
Hu, H., Wu, J., Li-Chan, E. C. Y., Zhu, L., Zhang, F., Xu, X., Fan, G., Wang, L.,
427
Huang, X., Pan, S. (2013). Effects of ultrasound on structural and physical
428
properties of soy protein isolate (SPI) dispersions. Food Hydrocolloids, 30,
429
647–655. https://doi.org/10.1016/j.foodhyd.2012.08.001
430
Jambrak, A. R., Mason, T. J., Lelas, V., Paniwnyk, L., & Herceg, S. (2014). Effect of
431
ultrasound treatment on particle size and molecular weight of whey proteins.
432
Journal
433
https://doi.org/10.1016/j.jfoodeng.2013.08.012
of
Food
Engineering,
121,
15–23.
434
Jiang, L., Wang, J., Li, Y., Wang, Z., Liang, J., Wang, R., Chen, Y., Ma, W., Qi, B.,
435
Zhang, M. (2014). Effects of ultrasound on the structure and physical
436
properties of black bean protein isolates. Food Research International, 62,
437
595–601. https://doi.org/10.1016/j.foodres.2014.04.022
438
Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe
439
method and its correlation with surface properties of proteins. Biochimica et
440
Biophysica Acta, 624, 13–20. https://doi.org/10.1016/0005-2795(80)90220-2
441
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the
21
442
head of bacteriophage T4. Nature, 227, 680–685.
443
Malik, M. A., Sharma, H. K., & Saini, C. S. (2017). High intensity ultrasound treatment
444
of protein isolate extracted from dephenolized sunflower meal: Effect on
445
physicochemical and functional properties. Ultrasonics Sonochemistry, 39,
446
511– https://doi.org/519. 10.1016/j.ultsonch.2017.05.026
447
Martínez-Velasco, A., Lobato-Calleros, C., Hernández-Rodríguez, B. E., Román-
448
Guerrero, A., Alvarez-Ramirez, J., Vernon-Carter, E. J. (2018). High intensity
449
ultrasound treatment of faba bean (Vicia faba L.) protein: Effect on surface
450
properties, foaming ability and structural changes. Ultrasonics Sonochemistry,
451
44, 97–105. https://doi.org/10.1016/j.ultsonch.2018.02.007
452
Márquez-Alvarez, L. R., Ocano-Higuera, V. M., Rodríguez-Felix, F., Ruiz-Cruz, S.,
453
Del-Toro-Sanchez, C. L., & Márquez-Ríos, E. (2015). Production and
454
functional evaluation of a protein concentrate from giant squid (Dosidicus
455
gigas) fins obtained by alkaline dissolution. Journal of Food Processing and
456
Preservation, 39, 2215–2224. https://doi.org/10.1111/jfpp.12466
457
Morales, R., Martínez, K. D., Ruiz-Henestrosa, V. M. P., & Pilosof, A. M. R. (2015).
458
Modification of foaming properties of soy protein isolate by high ultrasound
459
intensity: Particle size effect. Ultrasonics Sonochemistry, 26, 48–55.
460
https://doi.org/10.1016/j.ultsonch.2015.01.011
461
Murrieta-Martínez, C. L., Ezquerra-Brauer, J. M., Ocaño-Higuera, V. M., Cinco-
462
Moroyoqui, F. J., Torres-Arreola, W., Márquez-Ríos, E. (2015). Isolation and
463
partial characterization of myosin from jumbo squid mantle (Dosidicus gigas),
464
CyTA-Journal
of
Food,
13,
392–399.
22
465
https://doi.org/10.1080/19476337.2014.988183
466
Murrieta-Martínez, C. L., Ocaño-Higuera, V. M., Suárez-Jiménez, G. M., & Márquez
467
Ríos, E. (2016). Squid protein characteristics and their potential industrial
468
applications. Interciencia, 41, 520–525.
469
O’Sullivan, J. J., Park, M., Beevers, J., Greenwood, R. W., & Norton, I. T. (2017)
470
Applications of ultrasound for the functional modification of proteins and
471
nanoemulsion formation: A review. Food Hydrocolloids, 71, 299–310.
472
https://doi.org/10.1016/j.foodhyd.2016.12.037
473 474
Riddick, T. M. (1968). Control of Colloid Stability Through Zeta Potential. Wynnewood: Livingston Pub. Co.
475
Singh, A., Benjakul, S., & Kijroongrojana, K. (2018). Effect of ultrasonication on
476
physicochemical and foaming properties of squid ovary powder. Food
477
Hydrocolloids, 77, 286–296. https://doi.org/10.1016/j.foodhyd.2017.10.005
478
Stefanović, A. B., Jovanović, J. R., Dojčinović, M. B., Lević, S. M., Nedović, V. A.,
479
Bugarski, B. M., & Knežević-Jugović, Z.D. (2017). Effect of the controlled high-
480
intensity ultrasound on improving functionality and structural changes of egg
481
white
482
https://doi.org/10.1007/s11947-017-1884-5
proteins.
Food
Bioprocess
and
Technology,
10,
1224–1239.
483
Tan, M. C., Chin, N. L., Yusof, Y. A., Taip, F. S., Abdullah, J. (2015). Characterization
484
of improved foam aeration and rheological properties of ultrasonically treated
485
whey
486
https://doi.org/10.1016/j.idairyj.2014.09.013
487
protein
suspension.
International
Dairy
Journal,
43,
7–14.
Tolano-Villaverde, I. J., Ocaño-Higuera, V. M., Ezquerra-Brauer, J. M., Santos-
23
488
Sauceda, I., Santacruz-Ortega, H., Cárdenas-López, J. L., Rodríguez-
489
Olibarria, G., & Márquez-Ríos, E. (2018). Physicochemical characterization of
490
actomyosin–paramyosin from giant squid mantle (Dosidicus gigas). Journal of
491
the
492
https://doi.org/10.1002/jsfa.8653
Science
of
Food
and
Agriculture,
98,
1787-1793.
493
Xiong, T., Xiong, W., Ge, M., Xia, J., Li, B., Chen, Y. (2018). Effect of high intensity
494
ultrasound on structure and foaming properties of pea protein isolate. Food
495
Research
496
https://doi.org/10.1016/j.foodres.2018.04.044
International,
109,
260–267.
497
Xiong, W., Wang, Y., Zhang, C., Wan, J., Shah, B. R., Pei, Y., Zhou, B., Li, J., Li, B.
498
(2016). High intensity ultrasound modified ovalbumin: Structure, interface and
499
gelation
500
https://doi.org/10.1016/j.ultsonch.2016.01.014
501
properties.
Ultrasonics
Sonochemistry,
31,
302–309.
Xue, S., Xu, X., Shan, H., Wang, H., Yang, J., & Zhou, G. (2018). Effects of high-
502
intensity
ultrasound,
high-pressure
processing,
and
high-pressure
503
homogenization on the physicochemical and functional properties of
504
myofibrillar proteins. Innovative Food Science and Emerging Technologies, 45,
505
354–360. https://doi.org/10.1016/j.ifset.2017.12.007
506
Woyewoda, A. D., Shaw, S. J., Ke, P. J., & Burns B. G. (1986). Recommended
507
Laboratory Methods for Assessment of Fish Quality, Canadian Technical
508
Report of Fisheries and Aquatic Sciences No. 1448, Nova Scotia, Canada.
509
Table 1. Zeta potential of ultrasound-treated giant squid (Dosidicus gigas) proteins Treatment
Zeta Potential (mV)
C
-25.67a ± 0.84
20-1
-23.03b ± 1.19
20-3
-18.33c ± 0.32
20-5
-18.27c ± 0.75
40-1
-19.43c ± 0.70
40-3
-16.10d ± 0.62
40-5
-19.87c ± 0.83
Control, C; 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. Different superscripts indicate significant differences (p < 0.05). The values are the average of three replicas ± sd. 1 2
Figure captions Figure 1. Ultrasound-induced pH changes to giant squid (Dosidicus gigas) protein. The values are the average of three replicas ± sd. Figure 2. Electrophoretic profile of ultrasound-treated giant squid (Dosidicus gigas) protein under (a) denaturing, (b) reducing, and (c) native conditions. Lane std, marker; C, Control; lanes 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; lanes 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. Figure 3. Surface hydrophobicity (So) of giant squid (Dosidicus gigas) proteins treated by ultrasound. C, Control; 20%-1 min, 20-1; 20%-3 min, 20-3; 20%-5 min, 20-5; 40%-1 min, 40-1; 40%-3 min, 40-3; 40%-5 min, 40-5. The values are the average of three replicas ± sd. Figure 4. Rheological properties of ultrasound-treated giant squid (Dosidicus gigas) proteins: (a) viscosity, (b) shear strength versus shear rate, and (c) viscosity at a constant shear rate. , control; 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. Figure 5. Particle size of ultrasound-treated giant squid (Dosidicus gigas) proteins. C, Control; 20%-1 min, 20-1; 20%-3 min, 20-3; 20%-5 min, 20-5; 40%-1 min, 40-1; 40%-3 min, 40-3; 40%-5 min, 40-5. Figure 6. Foaming capacity of ultrasound-treated giant squid (Dosidicus gigas) proteins. The values are the average of three replicas ± sd. Figure 7. Foaming stability of giant squid (Dosidicus gigas) proteins treated by ultrasound at (a) 20% and (b) 40% amplitude. C, control; 20-1, 20-3 y 20-5 are samples treated with 20 % amplitude at 1, 3 and 5 min, respectively; 40-1, 40-3 y 40-5 are samples treated with 40 % amplitude at 1, 3 and 5 min, respectively. The values are the average of three replicas ± sd.
Figure 1
Figure 2a
Figure 2b
Figure 2c
Figure 3
Figure 4a
Figure 4b
Figure 4c
Figure 5
Figure 6
Figure 7a
Figure 7b
Highlights
•
Ultrasound altered the particle size and the 3D structure of the squid proteins
•
The rheological behavior was affected by the ultrasound treatment
•
Ultrasound-induced conformational changes to proteins improved the foaming property
AUTHOR DECLARATION
We wish to draw the attention of the Editor to the following facts which may be considered as potential conflicts of interest and to significant financial contributions to this work. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process. He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected].
Dr. Enrique Márquez Ríos, PhD. E-mail: [email protected] Main Researcher